Reaction mixture for use in isothermal amplification
View Patent ↗A method of monitoring amplification of a nucleic acid by providing a nucleic acid and an amplification mixture using the kit of isothermal reagents to a pH sensor or pH indicator, amplifying the nucleic acid using isothermal amplification, and detecting a change in pH due to the amplification using the pH sensor or pH indicator. The kit of reagents comprises a magnesium salt, a quaternary ammonium salt, and an alkali base.
1. A method of monitoring amplification of a nucleic acid comprising:
providing a nucleic acid in an amplification mixture comprising a magnesium salt, an alkali base, and at least one of: a quaternary ammonium salt, ammonium chloride, or guanidine hydrochloride, wherein the mixture has a buffer capacity less than 10 mM, to a pH sensor or pH indicator;
amplifying the nucleic acid using isothermal amplification; and
detecting a change in pH due to the amplification using the pH sensor or pH indicator.
2. The method of claim 1 , wherein the pH indicator is a colorimetric or fluorescent dye.
3. The method of claim 1 , wherein the pH sensor is an Ion Sensitive Field Effect Transistor (ISFET).
4. The method of claim 1 , further comprising:
determining a reaction time needed to change the pH of the mixture greater than a predetermined amount of change; and
quantifying a starting concentration of the nucleic acid based on said reaction time.
5. The method of claim 1 , wherein the mixture is in fluid communication with a reference electrode.
6. The method of claim 1 , wherein the mixture further comprises one or more allele specific primers having at least one base complementary to a target Single Nucleotide Polymorphism (SNP) of the nucleic acid, the method further comprising identifying said at least one base of the nucleic acid depending on whether amplification proceeds, as detected by the pH sensor or pH indicator.
7. The method of claim 1 , wherein the amplification changes the proton concentration of the mixture by more than 10% of the buffer capacity of the mixture.
8. The method of claim 1 , wherein the buffer capacity of the mixture is set to less than 5 mM.
9. The method of claim 1 , wherein the buffer capacity of the mixture is set to less than 1 mM.
10. The method of claim 1 , wherein the mixture further comprises a buffering agent.
11. The method of claim 10 , wherein the buffering agent is selected from the group consisting of Tris, HEPES, bicine, and MOPS.
12. The method of claim 10 , wherein the concentration of the buffering agent in the mixture is less than 5 mM.
13. The method of claim 1 , wherein the mixture further comprises sulphate compounds.
14. The method of claim 13 , wherein the concentration of the sulphate compounds in the mixture is less than 15 mM.
15. The method of claim 1 , wherein the concentration of the quaternary ammonium salt, ammonium chloride, or guanidine hydrochloride in the mixture is between 2 mM and 15 mM.
16. The method of claim 1 , wherein the concentration of the alkali base sets the pH of the mixture between 6 and 9.
17. The method of claim 1 , wherein the alkali base is one of NaOH, KOH, or LiOH.
18. The method of claim 1 , wherein the mixture further comprises one or more primers used in the amplification of the nucleic acid, which primers are allele specific such that amplification indicates the presence of a target nucleic acid.
19. The method of claim 1 , wherein the isothermal amplification is Strand Displacement amplification.
20. The method of claim 1 , wherein the isothermal amplification is Loop-mediated isothermal amplification (LAMP).
21. The method of claim 1 , wherein the buffering capacity of the mixture substantially masks the expected amount of protons released in the absence of amplification.
22. The method of claim 1 , wherein the mixture further comprises a strand displacement enzyme, nucleotides, and primers.